Infections caused by pathogenic species of Yersinia involve the integration of virulence activities that are tightly regulated. It is well established that both the etiologic agent of the plague, Y. pestis, and the enteropathogens, Y. enterocolitica and Y. pseudotuberculosis, each maintain the Ysc type III secretion (T3S) system that plays a critical role in pathogenesis. Yersinia utilize the Ysc T3S system to deliver a number of toxic Yop effector proteins directly into host cells, where they alter the host cell response to infection. Induction of the Ysc T3S system and secretion of the Yop effectors is regulated by environmental signals such as contact with a host cell, calcium- limitation and temperature. Transcriptional regulation of the Ysc T3S system and Yop effectors is under control of the AraC-like transcriptional regulator VirF/LcrF and its negative regulator YmoA, a bacterial histone-like protein. More recently, we have shown in Y. enterocolitica that the Ysc T3S system is part of a regulatory network involving the second messenger cAMP and CRP, a member of the cAMP receptor family of transcriptional regulators. The cAMP-CRP regulatory system affects Ysc T3S by a mechanism altering the transcription of virF. In the murine model of infection, Y. enterocolitica mutants blocked for cAMP synthesis or lacking CRP are severely attenuated, but induce protection against subsequent infection by fully virulent Y. enterocolitica. Thus, to further understand how the cAMP regulatory system controls Ysc T3S and to gain insight on the immune protection conferred by vaccination with cAMP-CRP regulatory mutants, we propose the following two specific aims. Three hypotheses will be tested in Aim 1: A.) cAMP levels change in response to environmental conditions that induce the Ysc T3S system; B.) The cAMP regulatory system affects expression of virF, the "master" regulator of the ysc-yop gene cascade; and C.) Histopathology analysis will reveal important differences in the host response to infection by wild type and cAMP regulatory mutants. Two hypotheses will be tested in Aim 2: A.) Expression of the Ysc T3S system and secretion of Yop effectors is affected by the cAMP regulatory system in Y. pestis; and B.) The cAMP regulatory system affects virulence of Y. pestis in the murine infection model. [unreadable] [unreadable] This research aims at understanding how species of Yersinia pathogenic to humans cause disease. The long-term goal of this research is to provide an understanding of fundamental mechanisms used by pathogenic Yersinia, and more generally pathogenic bacteria, to promote human infections and to conduct preliminary evaluations of measures that can be formulated into a scheme for disease prevention. [unreadable] [unreadable] [unreadable] [unreadable]